Journal: Oxidative Medicine and Cellular Longevity

Article Title: p66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke

doi: 10.1155/2018/5837123

Figure Lengend Snippet: Pharmacological inhibitors of PKC β and PKC δ suppressed CSE-induced p66Shc activation and its mitochondrial translocation in airway epithelial cells. The total and phosphorylated protein expression of PKC β and PKC δ was notably increased in Beas-2b cells exposed to 7.5% CSE for 24 h, and these alterations could be downregulated by 30 min pretreatments with 10 μ M LY333531 and 5 μ M rottlerin. A representative band is shown for each condition (a), and densitometric analysis of the bands was carried out by Bandscan 5.0 software to quantify the protein expression (b). Further, Western blot analysis revealed that the pretreatments with LY333531 and rottlerin significantly decreased the protein expression of p66Shc and p-p66Shc in the whole cell lysates (c) and suppressed p66Shc mitochondrial translocation and cytochrome c release from the mitochondria (d) in Beas-2b cells incubated with 7.5% CSE for 24 h. All the data are shown as the mean ± SD from three independent experiments. ∗ P < 0.05 versus the control group and # P < 0.05 versus the 7.5% CSE-treated group.

Article Snippet: The membranes were then incubated with specific antibodies against p66Shc (1 : 1000, Abcam), p-p66Shc (S36, 1 : 1000, Abcam), PKC β (1 : 1000, Proteintech Group), p-PKC β (T642, 1 : 800, Abcam), PKC δ (1 : 800, Proteintech Group), p-PKC δ (Y311, 1 : 3000, Abcam), cytochrome c (1 : 800, Proteintech Group), GAPDH (1 : 1000, Santa Cruz), and COXIV (1 : 600, Abcam) at 4°C overnight.

Techniques: Activation Assay, Translocation Assay, Expressing, Software, Western Blot, Incubation, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: p66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke

doi: 10.1155/2018/5837123

Figure Lengend Snippet: Pretreatments with PKC β and PKC δ inhibitors ameliorated CSE-induced mitochondrial damage of airway epithelial cells. Mitochondrial reactive oxygen species (ROS) was observed by confocal microscopy (a), and mitochondrial membrane potential (MMP) was determined by flow cytometry (b). Quantitative analysis showed that pretreatments with LY333531 and rottlerin reduced mitochondrial ROS content (c) and increased the levels of MMP (d) and intracellular ATP (e) in Bease-2b cells stimulated with 7.5% CSE for 24 h. All the results are shown as the mean ± SD from three independent experiments. ∗ P < 0.01 versus the control group and # P < 0.05 versus the 7.5% CSE-treated group.

Article Snippet: The membranes were then incubated with specific antibodies against p66Shc (1 : 1000, Abcam), p-p66Shc (S36, 1 : 1000, Abcam), PKC β (1 : 1000, Proteintech Group), p-PKC β (T642, 1 : 800, Abcam), PKC δ (1 : 800, Proteintech Group), p-PKC δ (Y311, 1 : 3000, Abcam), cytochrome c (1 : 800, Proteintech Group), GAPDH (1 : 1000, Santa Cruz), and COXIV (1 : 600, Abcam) at 4°C overnight.

Techniques: Confocal Microscopy, Membrane, Flow Cytometry, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: p66Shc Mediates Mitochondrial Dysfunction Dependent on PKC Activation in Airway Epithelial Cells Induced by Cigarette Smoke

doi: 10.1155/2018/5837123

Figure Lengend Snippet: Pretreatments with PKC β and PKC δ inhibitors attenuated CSE-induced airway epithelial cell injury. Cell apoptosis was determined by flow cytometry (a), and apoptosis rates of Beas-2b cells treated with 7.5% CSE and PKC β / δ inhibitors were both significantly decreased compared to the cells treated only with 7.5% CSE (b). Pretreatments with LY333531 and rottlerin significantly upregulated cell viability (c) and downregulated culture supernatant concentration of IL-6 and TNF- α (d) in Beas-2b cells exposed to 7.5% CSE. Data are expressed as the mean ± SD from three independent experiments. ∗ P < 0.01 versus the control group and # P < 0.05 versus the 7.5% CSE-treated group.

Article Snippet: The membranes were then incubated with specific antibodies against p66Shc (1 : 1000, Abcam), p-p66Shc (S36, 1 : 1000, Abcam), PKC β (1 : 1000, Proteintech Group), p-PKC β (T642, 1 : 800, Abcam), PKC δ (1 : 800, Proteintech Group), p-PKC δ (Y311, 1 : 3000, Abcam), cytochrome c (1 : 800, Proteintech Group), GAPDH (1 : 1000, Santa Cruz), and COXIV (1 : 600, Abcam) at 4°C overnight.

Techniques: Flow Cytometry, Concentration Assay, Control